Reagent Delivery by Partial Coalescence and Noncoalescence of

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Reagent Delivery by Partial Coalescence and Noncoalescence of Aqueous Microdroplets in Oil Carina S. Minardi, Mazdak Taghioskoui, Seong J. Jang, and Kaveh Jorabchi* Department of Chemistry, Georgetown University, Washington, D.C. 20057, United States S Supporting Information *

ABSTRACT: Reagent delivery constitutes a key step for reaction initiation in droplet-in-oil microfluidic platforms. Currently, this function is performed by complete fusion of a reagent droplet with the reactor droplet. The full coalescence, however, constrains the lower limit of volume delivery because reproducible droplet generation becomes exceedingly difficult as the reagent droplet volume is decreased. Here, we demonstrate fractional volume delivery based on partially coalescent and noncoalescent droplet collisions as a new reagent delivery mechanism. A charged reagent droplet is generated by pulsing a flow carrying needle to high voltage. The charged droplet is directed toward a grounded reactor droplet. Upon collision, the reagent droplet inverts its charge and is pulled away from the reactor droplet prior to full fusion, injecting only a fraction of its volume. The undelivered portion of the reagent drop is then merged with a collector droplet. We demonstrate that a wide range of fractional injections (0.003%−56%) can be reproducibly achieved, providing a means for minute volume delivery without small drop generation. ater microdroplets in immiscible fluids provide an isolated environment for chemical, biochemical, and biological investigations. Small volumes of microdroplets dramatically reduce the use of solvents, reagents, and precious materials such as enzymes. Moreover, microfluidic approaches can be devised for handling microdroplets, providing highthroughput platforms.1 These features have driven the rapid growth of droplet-based microfluidics in recent years with numerous reviews summarizing technical improvements and applications of microdroplets as reactors.2−7 More importantly, droplet-based microfluidics offer new experimental possibilities. For example, encapsulated single cells can be analyzed in droplets, revealing heterogeneities in cell populations and details of cellular functions.8 Interestingly, the small volume of a droplet can result in large cell densities for only a few encapsulated cells, providing a new approach for quorum sensing studies.9 Droplets can also act as artificial cells for investigation of biological functions.10 The processes in droplet-based microfluidics occur within three main stages: (1) reactor droplet generation, (2) reagent delivery to the droplet, and (3) analysis of droplet contents. Here, we primarily focus on the second stage. Reagent delivery is generally achieved through fusion (full coalescence) of reagent droplets with the reactor droplet. The fusion can occur spontaneously as the reagent and reactor droplets are brought together via slowing or trapping of one droplet in a train of droplets flowing in a channel (passive fusion).11−14 Because of its reliance on surface tension for fusion, this approach may encounter problems in applications where droplets are stabilized by surfactants. Alternatively, active fusion may be used to merge the droplets by electric,15−18 pneumatic,19,20 and

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laser21 activation commands. Active fusion provides better control over volume and timing of reagent delivery but requires more complex command circuitry and synchronization between reagent and reactor droplets. Minimizing change in reactor droplet volume upon reagent delivery is desirable to avoid dilution of product concentrations and to maximize detection efficiency. The dilution would be negligible when the reagent droplet volume is less than 1% of the reactor droplet volume. This necessitates generation and fusion of reagent droplets with 5-fold smaller diameters than that of the reactor droplet. The demand for small volume delivery is furthered by the emerging trends toward pico- and femtoliter reactors that offer fast diffusional mixing and novel experimental opportunities such as single molecule kinetic studies.22 Volume deliveries as low as 0.5 pL (10-μm diameter) have been reported in microfluidic devices.18 In efforts to push the limits lower, new mechanisms have been proposed for generation and fusion of droplets as small as 0.5 fL (1-μm diameter).22,23 These methods, however, rely on small orifices22 prone to clogging or jetting mechanisms23 that suffer from low reproducibility. Here, we report fractional volume injection as a new reagent delivery mechanism to alleviate the need for small droplet generation. Water droplets subjected to high electric fields in immiscible fluids make brief contact and separate from one another prior to full fusion.16,24−30 The underlying physical principles of this Received: April 10, 2013 Accepted: June 11, 2013

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200- or 350-cSt silicone oil (Xiameter PMX-200, Dow Corning, Austin, TX). A 5-μL reactor droplet and a 30-μL collector droplet were pipetted onto the droplet supports. Droplets included 500 nM isoleucine (Sigma Aldrich, St. Louis, MO) as internal standard and 5 mM ammonium acetate (Fisher Scientific, Watham, MA) as electrolyte. HPLC water (JT Baker, Austin, TX) was used as solvent for reagent, reactor, and collector droplets. The reactor droplet support was connected to the input of an oscilloscope (DS 1102E, Rigol, Oakwood Village, OH) while 650−1200 V was applied to the collector droplet using a highvoltage power supply (PS-350, Stanford Research System, Inc., Sunnyvale, CA). A second high voltage power supply (PS-350) connected to a voltage divider and a high-voltage relay (DAT70510, Cynergy3, Palatine, IL) controlled the reagent delivery needle voltage. An actuation pulse of 185−190 ms was applied to the relay by a pulse generator (801, Wavetek, San Diego, CA), which was in turn triggered by a frequency generator (PI-9587, Pasco Scientific, Hayward, CA) at 0.25 Hz. The reservoir was monitored by a digital camera (model DMK 41BU02.H, ImagingSource, Charlotte, NC) mounted on a stereo microscope throughout the experiments. A start-up stabilization period was used prior to reagent delivery. For this purpose, the collector droplet voltage was set to zero. The syringe pump was turned on and the needle voltage power supply was set to 800 V. This applied a fraction of the power supply voltage to the needle through the voltage divider. Upon triggering the relay, the full power supply voltage was applied to the needle emitting charged reagent droplets that were guided toward the collector droplet. Collection of 2− 3 droplets was sufficient to ensure syringe pump and droplet generation stability. For reagent delivery to reactor droplet, the collector and needle power supplies were set to optimized operating values listed in Table 1 after the initial stabilization period. These parameters resulted in generation of charged reagent droplets that followed the trajectory depicted in Figure 1, injecting a fraction of their volume into the reactor droplet. Details of reagent delivery are discussed below in the Results and Discussion section. Volume transfer from reagent droplet into the reactor droplet was characterized on the basis of the concentration of the analytes in the reagent solution, the volume of the reactor droplet (5 μL), and the concentration of analytes in the reactor droplet after reagent delivery. Reactor Droplet Analysis. Following reagent delivery, 4 μL of the reactor droplet was extracted from oil using a 10-μL syringe (Gastight #1701, Hamilton, Reno, NV). The extract was diluted with methanol for triplicate analysis by flow injection coupled to atmospheric pressure chemical ionizationtandem mass spectrometry (APCI-MS/MS). A syringe pump (P-500 Pharmacia Biotech) provided 5 mM ammonium acetate in 1:1 ACN:H2O at 200 μL/min for flow injection−APCI-MS/ MS analyses. A multiple reaction monitoring method was developed for analyte quantitation (m/z transitions: 166/120,

phenomenon have been investigated in recent studies using optical microscopy,24−30 identifying two major categories: (1) partial coalescence, where a discernible change in droplet diameters results from the contact between the droplets,26,28−30 and (2) noncoalescence, where no change in diameter of the droplets is detected after contact. 24,25 Thus far, the investigations have focused on characterizing partial coalescence and noncoalescence with the goal of identifying conditions that avoid these regimes. This goal stems from the desire for full coalescence in demulsifiers (used in separation of water from crude oil)31 and in droplet-based microfluidics. In contrast, we deliberately induce partial coalescence and noncoalescence to deliver a fraction of the reagent droplet volume into a reactor droplet. We investigate the potential of this new mechanism using nanoliter reagent droplets. The partial injection approach allows use of large reagent droplets to deliver a wide range of reagent volumes.

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EXPERIMENTAL SECTION Reagent Delivery. A schematic of the experimental setup is depicted in Figure 1. Two 2-56 stainless steel screws were

Figure 1. Schematic of the experimental setup for reagent delivery in partial coalescence and noncoalescence regimes. Charged reagent droplets are fired toward the reactor droplet and are pulled toward the collector droplet prior to full fusion. This results in partial volume injection by the reagent droplets.

inserted into a Teflon reservoir (22 mm × 10 mm × 12 mm) from opposite sides to support aqueous droplets in oil. A stainless steel needle (100 μm i.d., 300 μm o.d., AB Sciex) placed orthogonally to the droplet supports delivered the reagent solution into the reservoir at a flow rate (1−5 μL/min) controlled by a syringe pump (New Era Pump Systems, Inc., Farmingdale, NY). The reagent solution consisted of 1−5 mM valine, phenylalanine, and lysine (Sigma Aldrich, St. Louis, MO) in 5 mM ammonium acetate. The reservoir was filled with Table 1. Operating Parameters for Reagent Delivery needle voltage (V)

delivery regime partial coalescence noncoalescence

oil viscosity (cSt)

before pulse

during pulse

needle voltage pulse width (ms)

collector voltage (V)

needle protrusion into the reservoir (mm)

200

825−1000

1650−2000

185

1150

3

8

350

483

1450

190

675

3

7.5

B

distance between droplet supports (mm)

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147/86, 118/74, 132/84) using a triple-quadrupole mass spectrometer (Q-trap, AB Sciex). The method was validated by placing 5-μL droplets with known concentrations of analytes in oil for the typical duration of experiments (∼15 min) followed by measuring the concentration using APCI-MS/MS. This procedure also confirmed that analytes were not transferred from water droplets to oil in the time frame of experiments. Safety Considerations. The experiments require use of high-voltage power supplies and connections. Extreme care must be taken to avoid electrical shock.

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RESULTS AND DISCUSSION In the following, the partial and noncoalescence regimes are considered separately as they show drastically different behaviors for reagent delivery using the same reagent droplet size. Partial Coalescence. Video S-1 (Supporting Information) illustrates the reagent delivery into the reactor droplet in the partial coalescence regime using 200-cSt oil. Pulsing the needle to high voltage via relay closure results in creation of a nanoliter-sized charged reagent droplet. The electric field set by potentials on the needle and the collector guides the reagent droplet toward the reactor droplet. The reagent and the reactor droplets are polarized in the electric field. The needle has positive polarity, resulting in concentration of positive charges on the leading edge of the reagent droplet while negative ions concentrate in the trailing edge of the reagent droplet. As droplets approach each other, the oil film drains out of the contact area and a liquid bridge is formed between the two drops. The contents of the reagent droplet are then injected into the reactor droplet driven by the pressure imbalance between the two droplets29 and capillary forces.26 The rapid injection coupled with droplet polarization leads to transfer of more positive ions into the reactor droplet than negative ions, leaving a net negative charge on the reagent droplet.26 This results in attraction of the reagent droplet back toward the needle, separating the two droplets prior to full fusion. Electric fields of 1−5 kV/cm are generally needed to observe this behavior. Comparison of scaling analysis and experimental observations suggests that the extent of partial coalescence is determined by the balance between the Coulombic force pulling the charge-inverted reagent droplet and the surfacetension-driven inertia that pushes the droplet into the reactor.26 We monitor the timing of the droplet contact by tracking the voltage on the reactor droplet using an oscilloscope. The injected charge from the reagent droplet passes through the internal resistor of the oscilloscope and creates a voltage peak. The area underneath the peak reflects the amount of the injected charge. Figure 2 depicts a typical trace for timing of relay actuation and droplet discharge. The pulse duration for the relay actuation is optimized so that the needle voltage is returned to its low value ∼10 ms after reagent delivery (charge injection). Upon lowering the needle voltage, the collector droplet becomes more positive compared to the needle, attracting the undelivered portion of the reagent droplet. A large collector droplet is used so that multiple deliveries and collections can be performed with minimal effect on the volume of the collector. The change in the collector volume would result in a change in electric fields, impacting the degree of coalescence between the reagent and reactor droplets. Two parameters are investigated in the partial coalescence regime to

Figure 2. Typical timing diagrams for needle voltage, collector droplet voltage, and charge injection by the reagent droplet. The values correspond to an experiment with 133-nL reagent droplets in 200-cSt oil. The inset shows the charge injection trace captured by the oscilloscope with an area corresponding to 34 pC. Note that the oscilloscope was triggered by the charge injection trace for charge measurements to accurately capture the discharge curve.

control the injection volume: reagent droplet volume and electric field. Injection Tuning by Reagent Droplet Volume. The reagent droplet volume was controlled by the flow rate of the reagent solution at constant pulsing parameters. Flow rates of 1−5 μL/ min at droplet generation frequency of 0.25 Hz resulted in reagent droplets of 67−333 nL. Only one injection event was conducted prior to reactor droplet analysis. An average of three trials for each droplet size is reported in Figure 3. The injection

Figure 3. Effect of reagent droplet volume on injected volume in the partial coalescence regime. Reagent droplet volume is controlled by setting reagent solution flow in the 1−5 μL range at constant 0.25 Hz operation, 1650 V needle voltage, and 1150 V collector voltage. Other experimental conditions are listed in Table 1. Error bars represent standard deviations based on triplicate repetitions of injections at each setting. Linear fits: valine, y = 0.557x + 18.85, r2 = 0.979; lysine, y = 0.572x + 0.542, r2 = 0.993; phenylalanine, y = 0.579x + 7.29, r2 = 0.995.

volume shows a linear dependence on reagent droplet volume, implying that a constant fraction (56%) of the reagent droplet is injected into the reactor droplet. This fraction is the same for the three analytes, confirming that analyte transfer is achieved by volume transfer and that other factors such as interfacial affinity and ion mobility do not play a significant role in analyte transfer amount in this regime. This conclusion is also supported by our charge measurements. For example, 67-nL C

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reagent droplets inject ∼20 pC of charge into the reactor droplet under these conditions. Assuming all of the charge is carried by a single electrolyte of 5 mM concentration, a maximum equivalent injection volume of 0.02 pL would result. This volume is negligible in comparison to the measured injection volume using 67-nL reagent droplets (Figure 3). Larger droplets carry a larger amount of charge and move faster in the electric field. However, the injection percentage is not affected by the kinetics of the droplet motion because the capillary forces play the major role in partial coalescence.26 The injection volumes show increasing reproducibility as the reagent droplet volume decreases (RSD 5% and 15% for reagent droplet volumes of 66 and 333 nL, respectively). This effect may be explained by the lower stability of charged droplet generation for large droplets. Injection Tuning by Electric Field. Figure 4 depicts the effect of needle voltage on the fraction of 133-nL reagent droplets

collide and separate from one another with seemingly no change in the volume of the droplets.24,25 The transition from partial coalescence to noncoalescence occurs as the electric field surpasses a threshold. Noncoalescence is explained by a large negative curvature across the meniscus between the droplets upon contact, leading to a high pressure in the meniscus and an outward flow from the meniscus into the droplets. In other words, the contact conditions with elongated (polarized) droplets in high electric fields do not favor the growth of the meniscus and coalescence of the droplets. However, charge reversal of the moving droplet is observed in noncoalescence, similar to partial coalescence.24,25 This implies that material transfer is involved in noncoalescence and that the process may be suitable for reagent injection in microfluidic approaches. For reagent delivery in the noncoalescence regime two main questions must be answered: (1) What is the extent of material transfer in noncoalescence? (2) Is the injection amount a result of bulk volume transfer or are other transfer mechanisms involved in a significant manner? These questions are important because tunable bulk volume injection is desired in microfluidic approaches for predictable injection outcomes regardless of molecular properties of the reagents. Quantitative analysis of droplet volumes and contents is required to answer the questions above. The imaging methods rely on measurements of droplet radii and do not have the precision to quantify the very small material transfer in noncoalescence.24−26 In contrast, our mass spectrometric analyses allow measurement of droplet contents after reagent delivery, providing a quantitative method to characterize small material transfer. The jet formation at high electric fields limits our ability to reach noncoalescence regime using 200-cSt oil. Fortunately, higher viscosities promote the onset of the noncoalescence regime26 and suppress electrospray formation. Therefore, we utilized 350-cSt oil for noncoalescence studies, achieving stable droplet formation and reproducible noncoalescent collisions (see Table 1 for operating conditions). To maximize measurement precision and sensitivity, we used an averaging approach by performing 20−40 noncoalescent collisions prior to extracting and analyzing reactor droplet contents. Figure 5 demonstrates the injected fraction of reagent droplet volume per collision for three analytes in the noncoalescence regime. These values are calculated by measuring analyte concentrations in the reactor droplets after 40 collisions of 67-nL droplets and 20 collisions of 133-nL

Figure 4. Effect of needle voltage on fractional volume injection in partial coalescence regime using 133-nL reagent droplets. Error bars represent standard deviations based on triplicate repetitions of injections at each setting. Other experimental conditions are listed in Table 1. Linear fits: valine, y = −0.094x + 210, r2 = 0.998; lysine, y = −0.095x + 215, r2 = 0.972; phenylalanine, y = −0.089x + 203, r2 = 0.972.

injected into the reactor drop. At higher voltages, a higher attractive Coulombic force is exerted on the reagent droplet upon charge transfer with the reactor droplet, leading to a decrease in the amount of injected volume. The injection reproducibility is better at higher voltages in Figure 4. We have observed larger variations in droplet contact time for lower voltages compared to higher voltages, suggesting that instabilities in droplet generation at lower voltages may be the reason for the observed reproducibility trend. The upper limit for the needle voltage is dictated by a transition to jet formation (electrospray) from single droplet firing. Alternative droplet generation mechanisms such as a piezoelectric method are expected to alleviate these limitations. The overall injection reproducibility can be estimated by averaging relative standard deviations (RSD) in Figures 3 and 4. This yields average RSD values of 10.9%, 13.3%, and 11.3% for valine, lysine, and phenylalanine, respectively, demonstrating acceptable droplet-to-droplet reproducibility for volume delivery. Better reproducibility can be achieved by incorporating alternative droplet generation methods, as discussed above. Noncoalescence. In the recently reported noncoalescence phenomenon, oppositely charged droplets in an electric field

Figure 5. Injected fractions of reagent droplet volume in the noncoalescence regime using 67- and 133-nL droplets. The values correspond to 2 and 4 pL for 67- and 133-nL reagent droplets, respectively. Reported values are per collision based on three measurements with 40 collisions for 67-nL reagent droplets and 20 collisions for 133-nL droplets. Other experimental conditions are listed in Table 1. D

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droplets. Three findings can be inferred from Figure 5. First, the injected fraction is orders of magnitude lower than that in partial coalescence, leading to picoliter volume injections using nanoliter droplets. These results represent the first quantification of noncoalescent volume transfer, showing not only the exact amount but also the acceptable reproducibility of the injections. Second, the three analytes illustrate similar fractional injection values (0.003% of the reagent droplet volume). One may expect interfacial effects (surface sampling) to play a role in analyte transfer at such small fractional injections. Partitioning of amino acids to the oil−water interface with a variety of oils has been characterized, showing free energies of transfer from bulk water to the interface in −20 to −30 kJ/mol range.32 In addition, the amino acids used in our studies generally demonstrate 5−9 kJ/mol difference in their free energies of transfer.32 These values applied to our experimental conditions reveal that less than 1% of the total amount of each amino acid in the reagent droplet resides at the interface. Moreover, there would be a factor of 5−10 difference in concentrations of three amino acids at the interface. Absence of chemical fractionation in injection amounts, therefore, implies that interfacial sampling is negligible and that analytes are transferred mainly by bulk volume injection. The interfacial effects, however, may be observed for strongly adsorbed analytes (e.g., surfactants), where bulk volume concentration is depleted significantly to supply the analytes to the interface. Similar to partial coalescence, the amount of charge transfer in noncoalescence is negligible in comparison to the injected amounts of analytes, ruling out charge injection as the analyte transfer mechanism. Third, the transfer rate is not significantly affected by the reagent droplet volume, suggesting that volume delivery can be controlled by reagent droplet size, similar to the partial coalescence regime. This mode of volume delivery is under further investigation and will be presented in other reports dedicated to the noncoalescence regime.

electrocoalescence nature of this approach ensures applicability to surfactant-stabilized droplets, as demonstrated by full fusion methods. Our studies are conducted at a relatively low repetition rate to investigate the viability of fractional volume delivery. As evident in video S-1, droplet generation and collection of the undelivered portion of the droplet take the major part of each delivery cycle. The throughput, however, can be drastically improved in microfluidic platforms that offer short travel distances and fast droplet generation methods. An important factor for reactions in droplets is effective mixing of reagents upon delivery. In partial coalescence, a highvelocity jet of injected material is observed within the receiving droplet, which may improve mixing in small droplets.26 For thorough mixing, postdelivery mixers33 should be employed after partial coalescence and noncoalescence. This situation is not too different from full fusion. Although rapid mixing is observed in full coalescence of droplets with high surface tension,34−36 the lower surface tension in droplet-in-oil platforms as well as droplet−wall interactions in channels suppress the internal flows caused by coalescence, necessitating use of mixers.16,36−38

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ASSOCIATED CONTENT

S Supporting Information *

Video S-1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 202-687-2066. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Mr. Leon Der of the Physics Department at Georgetown University for his machine shop services. C.S.M. is grateful to the National Science Foundation Graduate Research Fellowship Program (DGE-0903443). S.J.J. would like to acknowledge the support of the American Chemical Society Project SEED for summer research.

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CONCLUSIONS AND IMPLICATIONS FOR MICROFLUIDIC PLATFORMS The results above demonstrate that fractional injection based on partially coalescent and noncoalescent collisions is a viable mechanism for reagent delivery into reactor droplets. The major advantage of this approach over full-fusion methods is that a wide range of volume deliveries (picoliter to nanoliter) can be achieved with relatively large (nanoliter) droplets, alleviating the complications of reproducible and robust small droplet generation in microfluidic applications. In our studies, partial coalescence provided fractional injections as low as 20%. Lower fractions were not reproducible, mainly due to electrospray formation during pulsed-voltage droplet generation. Alternative droplet generation schemes (e.g., pneumatic and piezoelectric generation) could provide lower fractions by eliminating the electrospray limitation. The noncoalescence regime offers delivery of extremely small volumetric fractions. We have demonstrated delivery of 0.003% of nanoliter-sized reagent droplets in the studies above. In our preliminary efforts to scale down this process, we have observed noncoalescent collisions between 50μm droplets generated by a piezoelectric device. Such approaches open a new avenue for delivery of attoliter and low femtoliter volumes for unique applications such as single cell analysis and single molecule kinetic studies. Moreover, the

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